Endocytosis includes distinct processes by which receptors, pathogens, nutrients, and environmental cues internalize into cells. Despite the ubiquity of endocytosis, its mechanism with respect to many putative biologics and drug carriers remains underexplored. To develop a toolkit for studying endocytosis, we harness ‘protein-polymers’ derived from elastin-like polypeptides (ELPs). When expressed in eukaryotes and heated, ELPs phase separate into cellular microdomains within minutes. We recently reported that ELP fusions with the epidermal growth factor receptor (EGFR) undergo temperature-mediated internalization to early endosomes, which activates downstream kinases. Having demonstrated that  membrane-associated EGFR fusions form microdomains, we here report a panel of ELP fusions designed to study endocytosis, which includes fusions to clathrin-light chain, caveolin-1, flotillin-1, and dynamin-2. In comparison with temperature-insensitive controls, each of these fusions phase separates at 37 °C. Fusions were characterized using fixed-cell confocal scanning laser microscopy and also live-cell epifluorescence imaging. We developed a novel strategy to label intracellular ELP microdomains using co-transfection with a GFP-ELP reporter protein. Notable differences were observed between cytosolic clathrin and membrane-associated caveolin. Cytosolic clathrin microdomains associate with and deplete endogenous proteins required for clathrin-mediated internalization, which blocks clathrin-mediated internalization above the transition temperature. In contrast, membrane-associated caveolin fusions rapidly internalize into cells upon heating. Caveolin assembly actively pulls cholera toxin subunit B, which is a marker for lipid rafts, from the cell surface into ELP microdomains. Thus for the caveolin fusions, ELP-mediated assembly initiates a process like caveolin-mediated endocytosis. Clathrin, caveolin, and flotillin-mediated endocytosis have all been reported as dynamin-dependent; furthermore, ELP-mediated phase separation of dynamin fusions promotes the internalization of cellular compartments of various sizes. We conclude that ELP fusions can trigger assembly of key proteins involved with different mechanisms of endocytosis. With further development, this toolkit may become a powerful strategy to explore the mechanisms of cellular internalization for chimeric receptors, antigens, pathogens, biological drugs, and drug carriers relevant to biology and medicine.

Cellular expression of temperature-dependent ELP microdomains modulates clathrin, caveolin, flotillin, and dynamin-dependent endocytosis

2019 Spring National Meeting

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